High-resolution Hadamard transform microscope fluorescence imaging: quantifying the DNA content in single cells

This study presents a novel miniaturized Hadamard transform fluorescence imaging microscope, by combining a conventional fluorescence microscope with Hadamard transform multiplexing encoding using a one-dimensional movable mask to realize spatial resolution and a linear CCD for multichannel detection. The microscope can provide high-resolution automatically-generated 0–255 gray level HT images for morphological analysis and visualization of a single cell, and normalized HT images for cellular quantitative measurements. The microscopes imaging capability was applied to measure the DNA content in human lymphocyte, chicken erythrocyte and eel erythrocyte, and a comparative study was performed. The results show that the calibrated DNA content in a chicken erythrocyte is 2.32 pg when human lymphocyte is used as the standard, and eel erythrocyte may be a potentially reliable and novel standard for determining DNA contents in other species because it has a stable DNA value of 2.06 pg, with a CV of 4.3% when 20 eel erythrocytes are measured. The results also demonstrate that the HT imaging microscope should be valuable in the fields of medicine and cell biology.     

高分辨阿达玛变换显微荧光图像分析(Ⅲ)——系统分辨率和图像恢复过程对单细胞分析结果的影响

提出了一种改进的阿达玛变换(HT)显微荧光图像分析系统,以单细胞试样分析为基础,分别对系统的分辨率和解码后的图像恢复过程进行了讨论.结果表明,该系统可应用于单细胞形态分析和定量分析.图像在x和y方向的像素分辨率相同,并达到了同一成像物镜下的空间分辨率水平,因此在获取微米级单细胞试样的微弱荧光信号的二维图像时,系统的成像能力较好,可用于单细胞形态分析.对花粉细胞的荧光衰退过程的定量分析结果表明,对不同HT图像提供的同一系列试样的定量数据进行比较时,必须对所有该系列试样的图像恢复过程进行归一化处理.     

微流控进样阿达玛变换显微荧光成像单细胞分析

将微流控芯片用于单细胞进样,以自制阿达玛变换显微荧光成像分析系统进行成像检测,讨论了影响单细胞进样和荧光成像的主要因素,并对花粉细胞DNA含量进行定量分析。结果表明：微流控进样技术与HT显微成像技术相结合,可有效可靠地应用于单细胞分析中,所测定的三个玉帘花粉的DNA含量分别为124．4、123．9和62．9pg。     

Hadamard transform fluorescence image microscopy using one-dimensional movable mask

With a 511-slit one-dimensional (1D) Hadamard mask and a highly sensitive linear charge-coupled device (CCD), spatial multiplexing is performed and a programmable Hadamard transform (HT) microscopic fluorescence imaging system was developed. The system can generate 511×512 pixel format images for small samples. Sensitivity, signal to noise ratio, imaging speed and spatial resolution of this system were discussed. The results show that the system can be applied for single-cell imaging sensitively in a short time. Spatial resolution up to 0.24 μm/pixel, which is close to the resolution limit of the conventional optical microscope, has been obtained under oil lens. The weak native fluorescence imaging for pollen cells can be realized within 1 min. The system has been applied for multi-parameter evaluation of tumor malignancy based on nuclear DNA ploidy measurements for one breast tumor specimen. The result indicates that the system has good application prospect in cell biology and medicine.     

高分辨阿达玛变换显微荧光图像分析(Ⅰ)——系统的成像能力及其在单个肿瘤细胞分析中的应用

阿达玛变换(Hadamard transform, HT)是一种类似于傅里叶变换的光谱调制技术, 具有多通道同时检测和多通道成像能力. 实现高分辨HT成像的关键在于阿达玛模板的制作, 阿达玛模板有两种, 即移动式机械编码模板(Movable mechanical mask)和固定式光电模板(Stationary electro-optic mask). 在实际成像方面, 移动模板和固定模板各有优缺点: 前者一般用石英玻璃制作, 对光信号不会因模板吸收而导致信号损失, 因此数据很可靠, 而且模板的制作也较为容易, 但由于采用步进电机驱动而容易导致机械故障, 难以实现快速编码; 后者无移动部件, 无机械故障, 因此系统比较紧凑, 但由于它是由液晶材料制成的(可导致信号损失), 从而限制了其在某些光谱区域的使用. 此外, 它对系统的软件设计要求比前者高, 实现高分辨成像更加困难. 正是由于上述原因, 实现快速、高分辨HT成像具有一定难度, 最近有关HT成像技术的报道极少.     

高分辨阿达玛变换显微荧光图像分析(Ⅱ)单细胞DNA荧光图像生成、处理及自动定量分析研究

本研究把荧光显微镜，阿达玛变换多通道成像技术和计算机图象处理技术结合起来，建立了一种单细胞DNA荧光图像自动定量分析系统，对系统的成像原理、信号编码和解码方法及单细胞定量分析方法进行了讨论，分析结果表明，本系统提供的单细胞定量分析数据稳定可靠。     

Ultra-thin temperature controllable microwell array chip for continuous real-time high-resolution imaging of living single cells

Single-cell imaging, a powerful analytical method to study single-cell behavior, such as gene expression and protein profiling, provides an essential basis for modern medical diagnosis. The coding and localization function of microfluidic chips has been developed and applied in living single-cell imaging in recent years. Simultaneously, chip-based living single-cell imaging is also limited by complicated trapping steps, low cell utilization, and difficult high-resolution imaging. To solve these problems, an ultra-thin temperature-controllable microwell array chip (UTCMA chip) was designed to develop a living single-cell workstation in this study for continuous on-chip culture and real-time high-resolution imaging of living single cells. The chip-based on ultra-thin ITO glass is highly matched with an inverted microscope (or confocal microscope) with a high magnification objective (100 × oil lens), and the temperature of the chip can be controlled by combining it with a home-made temperature control device. High-throughput single-cell patterning is realized in one step when the microwell array on the chip uses hydrophilic glass as the substrate and hydrophobic SU-8 photoresist as the wall. The cell utilization rate, single-cell capture rate, and microwell occupancy rate are all close to 100% in the microwell array. This method will be useful in rare single-cell research, extending its application in the biological and medical-related fields, such as early diagnosis of disease, personalized therapy, and research-based on single-cell analysis.     

High-throughput single-cell quantification using simple microwell-based cell docking and programmable time-course live-cell imaging

Extracting single-cell information during cellular responses to external signals in a high-throughput manner is an essential step for quantitative single-cell analyses. Here, we have developed a simple yet robust microfluidic platform for measuring time-course single-cell response on a large scale. Our method combines a simple microwell-based cell docking process inside a patterned microfluidic channel, with programmable time-course live-cell imaging and software-aided fluorescent image processing. The budding yeast, Saccharomyces cerevisiae (S. cerevisiae), cells were individually captured in microwells by multiple sweeping processes, in which a cell-containing solution plug was actively migrating back and forth several times by a finger-pressure induced receding meniscus. To optimize cell docking efficiency while minimizing unnecessary flooding in subsequent steps, circular microwells of various channel dimensions (4-24 μm diameter, 8 μm depth) along with different densities of cell solution (1.5-6.0 × 10(9) cells per mL) were tested. It was found that the microwells of 8 μm diameter and 8 μm depth allowed for an optimal docking efficiency (>90%) without notable flooding issues. For quantitative single-cell analysis, time-course (time interval 15 minute, for 2 hours) fluorescent images of the cells stimulated by mating pheromone were captured using computerized fluorescence microscope and the captured images were processed using a commercially available image processing software. Here, real-time cellular responses of the mating MAPK pathway were monitored at various concentrations (1 nM-100 μM) of mating pheromone at single-cell resolution, revealing that individual cells in the population showed non-uniform signaling response kinetics.     

Macromolecular contrast agents for optical imaging of tumors: comparison of indotricarbocyanine-labeled human serum albumin and transferrin

Macromolecules accumulate in solid tumors and can thus be used as carriers for the delivery of attached contrast agents to tumors. We report the synthesis and use of serum protein-dye conjugates consisting of transferrin (Tf) or human serum albumin (HSA) and an indotricarbocyanine (ITCC) derivative as contrast agents for the optical imaging of tumors. The compounds were characterized with respect to their photophysical properties and tested in vitro for their ability to bind to tumor cells and in vivo for their potential to delineate experimental tumors. In contrast to HAS-ITTC, Tf-ITCC showed receptor-mediated uptake by HT29 human colon cancer cells in vitro. After intravenous injection into HT29 tumor-bearing nude mice both compounds induced increased fluorescence contrast of tumors in vivo. After 24 h the contrast between tumor and normal tissue was significantly higher for Tf-ITCC than for HAS-ITCC. Dye-induced fluorescence was found to be predominantly located in perinecrotic areas of the tumor. Furthermore, Tf-ITCC produced fluorescence of viable tumor cells, whereas HAS-ITCC fluorescence was recorded along connective tissue. We conclude that ITCC-labeled Tf and HSA can serve as macromolecular contrast agents for the optical imaging of tumors, with Tf-ITCC showing higher efficiency.     

阿达玛变换显微荧光成像在单细胞定量分析中的应用研究

本文探讨了用阿达玛变换（HT）显微荧光成像系统获得基于256灰度级图像的单细胞荧光强度和解码定标值之间的关系,将归一化的HT图像用于定量分析,建立了适合不同样品的定量分析方法.在此条件下,定量分析数据有很好的准确度和精密度;将系统用于单个乳腺肿瘤细胞的DNA定量分析,对乳腺肿瘤的良恶性及癌变程度进行判断,分析结果与病理学诊断结论一致.     

Microfluidically-unified cell culture, sample preparation, imaging and flow cytometry for measurement of cell signaling pathways with single cell resolution

We have developed a microfluidic platform that enables, in one experiment, monitoring of signaling events spanning multiple time-scales and cellular locations through seamless integration of cell culture, stimulation and preparation with downstream analysis. A combination of two single-cell resolution techniques-on-chip multi-color flow cytometry and fluorescence imaging provides multiplexed and orthogonal data on cellular events. Automated, microfluidic operation allows quantitatively- and temporally-precise dosing leading to fine time-resolution and improved reproducibility of measurements. The platform was used to profile the toll-like receptor (TLR4) pathway in macrophages challenged with lipopolysaccharide (LPS)-beginning with TLR4 receptor activation by LPS, through intracellular MAPK signaling, RelA/p65 translocation in real time, to TNF-α cytokine production, all in one small macrophage population (< 5000 cells) while using minute reagent volume (540 nL/condition). The platform is easily adaptable to many cell types including primary cells and provides a generic platform for profiling signaling pathways.     

Capillary sodium dodecyl sulfate-DALT electrophoresis of proteins in a single human cancer cell.

Capillary Sodium dodlecyl sulfate (SDS)-DALT an (abbreviation for Dalton) electrophoresis was applied to analysis of proteins in single HT29 human colon adenocarcinoma cells. A vacuum pulse was employed to introduce a single cell into the coated capillary. Once the cell was lysed, proteins were denatured with SDS, fluorescantly labeled with 3-(2-furoyl)-quinoline-2-carboxaldehyde (FQ), and then separated by using 8% pullulan as the sieving matrix. This method offers a few advantages for single-cell protein analysis. First, it provides reproducible separation of single-cell proteins according to their size. Based on comparison with the migration time of standard proteins, most components from a single HT29 cancer cell have molecular masses within the range of 10-100 kDa. Second, as a one-dimensional separation method, it gives fairly good resolution for proteins. Typically, around 30 protein components of a single HT29 cell were resolved, indicating that this method has similar peak capacity to SDS-polyacrylamide gel electrophoresis (PAGE). Third, this method shows high detection sensitivity and wide dynamic range, which is important because of the wide range of protein expression in living systems. Detection limits for standard proteins ranged from 10(-10) to 10(-11) M. Finally, this method provides much higher speed than classical gel electrophoresis methods, and it provides automated anlysis of cellular proteins at the single-cell level; the separation is complete in 30 min and the entire analysis takes approximately 45 min.     

用于细胞核成像的2,7-BHVC双光子荧光探针

分别合成了2,7-双(1-羟乙基-4-乙烯基吡啶) 咔唑碘盐(2,7-BHVC)与3,6-BHVC, 进行了荧光性能和细胞核成像测试, 结果表明, 2,7位的Ф×δ是3,6位的2.8~3.6倍, 染色实验也证实了2,7-BHVC有特定成像细胞核的能力.     

Pump-probe optical microscopy for imaging nonfluorescent chromophores

Many chromophores absorb light intensely but have undetectable fluorescence. Hence microscopy techniques other than fluorescence are highly desirable for imaging these chromophores inside live cells, tissues, and organisms. The recently developed pump-probe optical microscopy techniques provide fluorescence-free contrast mechanisms by employing several fundamental light-molecule interactions including excited state absorption, stimulated emission, ground state depletion, and the photothermal effect. By using the pump pulse to excite molecules and the subsequent probe pulse to interrogate the created transient states on a laser scanning microscope, pump-probe microscopy offers imaging capability with high sensitivity and specificity toward nonfluorescent chromophores. Single-molecule sensitivity has even been demonstrated. Here we review and summarize the underlying principles of this emerging class of molecular imaging techniques.     

A cyanine based fluorophore emitting both single photon near-infrared fluorescence and two-photon deep red fluorescence in aqueous solution

Optical imaging provides an indispensable way to locate tumors in their early stages with high sensitivity and signal to background ratio. A heptamethine cyanine based fluorophore that emits both single photon near-infrared fluorescence and two-photon deep red fluorescence under physiological conditions was developed. Linear and nonlinear photophysical properties of this fluorophore were investigated and it demonstrated the capability to label lysosomes in cancer cells. The advantages of this fluorophore, including tolerable cytotoxicity, high fluorescence quantum yield, and the ability to emit both near-infrared single photon fluorescence and deep red two photon fluorescence in aqueous solution, give it potential to be used in intra-operatively optical image-guided tumor excision followed by two-photon fluorescence microscopy biopsy analysis after a single administration.     

Time-domain fluorescence lifetime imaging for intracellular pH sensing in living tissues

pH sensing in living cells represents one of the most prominent topics in biochemistry and physiology. In this study we performed one-photon and two-photon time-domain fluorescence lifetime imaging with a laser-scanning microscope using the time-correlated single-photon counting technique for imaging intracellular pH levels. The suitability of different commercial fluorescence dyes for lifetime-based pH sensing is discussed on the basis of in vitro as well of in situ measurements. Although the tested dyes are suitable for intensity-based ratiometric measurements, for lifetime-based techniques in the time-domain so far only BCECF seems to meet the requirements of reliable intracellular pH recordings in living cells.     

Probing hydroxyl radicals and their imaging in living cells by use of FAM-DNA-Au nanoparticles

The incorporation of gold nanoparticles (Au NPs) as quencher modules in fluorescent probes for DNA damage caused by intracellular hydroxyl radicals (HO*) is reported. Au NPs of 15 nm diameter were decorated with DNA oligomers terminating in thiol functions in their 3' positions and possessing 5' fluorophore modifications. The Au NPs, which have high extinction coefficients, functioned as excellent fluorescent quenchers in the fluorophore-Au NP composites. FRET is switched off as a factor of HO*-induced strand breakage in the single-stranded DNAs, restoring the fluorescence of the quenched fluorophores, which can be followed by spectrofluorimetry. In vitro assays with HO*-generating Fenton reagent demonstrated increases in fluorescence intensity with a linear range from 8.0 nM to 1.0 microM and a detection limit as low as 2.4 nM. Confocal microscopic imaging of macrophages and HepG2 revealed that the probe is cell-permeable and intracellular HO*-responsive. The unique combination of good selectivity and high sensitivity establishes the potential value of the probe for facilitating investigations of HO*-mediated cellular homeostasis and injury.     

High-intensity fluorescence imaging and sensitive electrochemical detection of cancer cells by using an extracellular supramolecular reticular DNA-quantum dot sheath

Acting as a cage-type cellular probe, an extracellular supramolecular reticular DNA-quantum dot (QD) sheath has been developed for high-intensity fluorescence microscopy imaging and the sensitive electrochemical detection of Ramos cells. The extracellular supramolecular reticular DNA-QD sheath is constructed from layer-by-layer self-assembly of DNA-CdTe QD probes and DNA nanowire frameworks functionalized with a Ramos cell-binding aptamer. The DNA-QD sheath forms specifically and quickly on the surface of Ramos cells at physiological temperature, and the assembly of large numbers of DNA-CdTe QD probes on the surface of Ramos cells produces exceedingly high fluorescence intensity. Using the extracellular supramolecular reticular DNA-QD sheath as the cellular probe, Ramos cells can be clearly observed and easily distinguished from a mixture of multiple cancer cells by fluorescence microscopy imaging. Using the new cage-type cellular probe, a sensitive sandwich-type electrochemical strategy has also been developed to achieve accurate quantitative analysis of Ramos cells. Under the optimized conditions, Ramos cells can be detected quantitatively in a range from 10 to 1000 cells with a detection limit of 10 cells. This strategy presents a promising platform for highly sensitive and convenient evaluation of cancer cell levels.